Research ArticleNEUROSCIENCE

A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity

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Science Advances  29 May 2020:
Vol. 6, no. 22, eaba0365
DOI: 10.1126/sciadv.aba0365
  • Fig. 1 A rapid method combining depigmentation with tissue clearing in representatives of four distinct and species-rich animal clades.

    (A) Main steps of the DEEP-Clear protocol, including incubation times for the five main model systems presented in this study. (B) Systematic advancement of eye depigmentation speed by acetone pretreatment in immature and mature worms. Quantified comparisons between acetone-treated (AcT) and untreated (UT) head halves incubated with Solution-1. All values are mean ± SD; statistical significance was determined by a Wilcoxon test, yielding P values of P = 0.00166 (immature worms) and P = 0.00192 (mature worms). (C) Systematic advancement of eye depigmentation speed by acetone pretreatment in squid. Quantification of depigmentation time in acetone-treated and untreated squid halves upon incubation with Solution- 1.1. Values are mean ± SD; statistical significance was determined by a Wilcoxon test (P = 0.01285). (D) Differential and synergistic impact of acetone, peroxide, and Solution-1.1 on zebrafish fin pigments. Panels show fins of untreated (top) and treated (bottom) zebrafish fins. Insets: Magnification of dashed area and impact of different treatments on respective pigments (black arrows). Xanthophore containing pteridine and carotenoid pigments (yellow and orange) and melanophore containing melanin pigment (black). Rightmost panels show the overall impact of the full DEEP-Clear protocol. (E) Wide-field images of specimens placed on top of a USAF 1951 chart. Uncleared samples in PBS (top panels), same samples after depigmentation and refractive index (RI) matching in Solution-2 (middle panels), and higher magnification of red rectangular areas indicating the highest level of transparency reached after RI matching (bottom panels). Scale bars in the insets of (D), 20 μm. In (A), dagger indicates the possibility of fixation with Bouin’s solution; asterisks indicate the use of Solution-1.1 incubation instead of Solution-1. o.n., overnight; RT, room temperature; h, hour; ’, minutes. In (B) and (C), *P < 0.05 and **P < 0.01. Photo credit: Marko Pende, Medical University of Vienna.

  • Fig. 2 Global and focused insight into the adult annelid nervous system.

    (A) Comparison of uncleared (left) and DEEP-Clear processed (right) bristle worm specimens. (B) Imaging of EGFP signal in photoreceptors of DEEP-Clear–processed pMos{rops::egfp}vbci2 animals by light-sheet microscopy. Arrowheads indicate projections from the eyes into the central-brain neuropil. Inset shows the XZ projection of the boxed area. (C and D) EGFP+ parapodial cell bodies and their projections (arrowheads) into and along the ventral nerve cord as visualized by light-sheet microscopy. (E to G) Anti-acetylated alpha-tubulin (anti-AcTub) immunolabeling revealing the annelid nervous system, visualized either by light-sheet microscopy [(E) whole animal, including the stereotypical trunk nervous system; inset showing major structures of the brain] or confocal microscopy [(F) anterior eye, with photoreceptor cells exhibiting segmentation into outer segments protruding into the central filling mass and basal cell bodies; (G) brain, with the inset showing a magnification of the boxed region of the anterior eye]. The specimen in (G) shows a colabeling by anti-Vasotocin (anti-VT) immunohistochemistry, and arrowheads indicate VT+ puncta (putative dense core vesicles) in deep neurite projections. Corresponding VT+ cell bodies can be seen in close proximity to the anterior eyes. an, antennal nerve; cb, cell body; fm, filling mass; no, nuchal organ; np, neuropil; os, outer segment; pac, parapodial receptor cells; ae, anterior eye; pe, posterior eye; prc, eye photoreceptor cell; sn.II, segmental nerve II; vnc, ventral nerve cord.

  • Fig. 3 Insight into the nervous system structures in whole-mount specimens of squid hatchlings.

    (A) Comparison of uncleared (left) and DEEP-Clear–processed (right) squid hatchlings (anterior up), documenting the removal of eye and body pigments (asterisk indicates remaining ink sac melanin). (B) Light-sheet acquisition of anti–phosphohistone H3 immunolabeling (bobtail squid, dorsal view, anterior to the right; dashed lines and box demarcate the area of the XZ slice shown in inset 1 and the enlargement in inset 2). DAPI, 4′,6-diamidino-2-phenylindole. (C to E) Visualization of the nervous system using anti-AcTub labeling. (C) Light-sheet acquisition (dorsal views, anterior up) in longfin inshore squid (left) and the Hawaiian bobtail squid (right). Inset shows two XZ projections of the respective areas (1 and 2) dashed in the XY view. (D and E) Confocal views of the deep medulla of the bobtail squid optic lobe, revealing the diagnostic structure of thick fiber tracts embracing perikarya (asterisks) and fine transversal fibers (arrowheads) of the Hawaiian bobtail squid. a, anterior; bcr, brachial crown; brn, brachial nerves; ceg, cerebral ganglion; opl, optic lobe; p, posterior; pan, pallial nerve; ret., retina; sbl, superior buccal lobe; stg, stellate ganglion; tft, thick fiber tract.

  • Fig. 4 DEEP-Clear–enabled analysis of the eyes and cranial nervous system of postlarval zebrafish.

    (A) Comparison of uncleared (left) and DEEP-Clear–processed (right) zebrafish juveniles. (B and C) Visualization of the nervous system using anti-AcTub immunohistochemistry (lateral views, anterior to right). (B) Light-sheet acquisition of central and peripheral nervous system (arrowheads indicating lateral line), with the right inset showing a close-up of the head. Left inset: confocal view of retina and bundles of retinal ganglion cells (arrowheads) exiting through the optic disk to form the optic nerve. (C) Light-sheet acquisition of a specimen colabeled by anti-serotonin immunohistochemistry. Magnifications in the top insets show serotonergic cells in (1) the spinal cord and (2) in the eye. The lower image shows a sagittal view of the head and a corresponding YZ slice (inset), with an asterisk close to serotonergic cells of the raphe area. (D) Sagittal view of a light-sheet–acquired juvenile head showing PH3+ cells. Lower insets show XZ projections of the indicated areas, revealing PH3-labeled cells deep in the brain, inset (1) provides an enlarged view of the right eye. All, anterior lateral line; hma, hyomandibular arch; soa, supraorbital arch; tec, tectum; trg, trigeminal ganglion; olf, olfactory epithelium; on, optic nerve; pll, posterior lateral line. Photo credit: Marko Pende, Medical University of Vienna.

  • Fig. 5 Whole-body immunolabeling of the juvenile axolotl nervous system.

    (A) Comparison of uncleared (left) and DEEP-Clear–processed (right) axolotl juveniles (around 3 months of age). (B to E) DEEP-Clear–processed juveniles stained by anti–beta III tubulin (anti-TUBB3) to mark the nervous system. (B) Overview of the juvenile anatomy and nervous system in a DEEP-Clear–processed specimen imaged with light-sheet microscopy. (C) Close-up of the head region, revealing the major brain neuroanatomy and branches of the cranial nerves. The inset shows three XZ projections taken at the indicated positions. (D) Focus on the eye region of a specimen revealing the projection of retinal ganglion cells (arrows) through the optic disk to form the optic nerve. (E) Axolotl nose showing innervation of the olfactory nerve. (F) Labeling of central and peripheral nervous system using anti-MBP antibody and 4′,6-diamidino-2-phenylindole. Inset shows higher magnification of peripheral nerves. nc, nasal cavity; oln, olfactory nerve; od, optic disk; Rhc, rhombencephalon; Mec, mesencephalon; Dic, diencephalon; C.N., cranial nerve. Photo credit: Marko Pende, Medical University of Vienna

  • Fig. 6 Retinal growth patterns and molecular signatures of annelid eye photoreceptors.

    (A to C) Codetection of incorporated EdU (magenta), riboprobes against ropsin-1 (overlay with EGFP is yellow, and pure signal is red), and EGFP (green) in premature pMos{rops::egfp}vbci2 bristle worms. Single and double asterisks indicate regions of cell proliferation in the anterior and posterior ganglionic region, respectively. (D and E) Similar codetection of EdU (magenta), ropsin-1 (red), and EGFP epitopes (green), including close-ups of the posterior (D, left) and anterior (E) eye region. Arrows point to the proliferative cells in either eye. (F) Fluorescent detection of riboprobes against ropsin-1 (left), gq (middle), and tmdc/c2433 (right) in comparative RNA whole-mount hybridizations, revealing expression of all three genes in eye photoreceptors of the worm head. All dorsal views, anterior to the top. ae, anterior eye; pe, posterior eye.

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